A crucial element in the development of effective prophylactic strategies for AIDS is an experimental animal model in which the course of immunodeficiency virus infection parallels the pathogenesis of the human disease. SIV infection of macaques is a relevant model since it induces an immunodeficiency syndrome in infected macaques that is remarkably similar to human AIDS. In an earlier vaccine effort, we evaluated the highly attenuated vaccinia virus Ankara (MVA) strain as a recombinant vector. MVA-SIV recombinants expressing Env, Gag-pol or both antigens were evaluated. Immunization with MVA-SIV recombinants resulted in a reduction in setpoint plasma viral load that was associated with prolonged survival. We then evaluated the effect of vaccination of memory CD4+ T cells in the blood;vaccination was associated with better preservation of memory CD4+ T cells and inversely correlated with virus load. Importantly, preservation of CD4+ T cells early post challenge correlated with improved survival and with titers of neutralizing antibodies to the challenge virus prior to challenge. Our previous vaccine studies have used SIVsmH4 as an immunogen;this virus has a lab-adapted, neutralization-sensitive envelope. Therefore, these studies focused mostly on generating cellular immune responses. The purpose of our current vaccine studies has been to use a prime-boost strategy to generate antibody that will neutralize the pathogenic SIVsmE543 and study the role of this antibody in the SIV model. Prior studies have only demonstrated a role for neutralizing antibody in vivo using the less pathogenic SIVmne model or in SHIV. Similar studies using SIVmac239 have been hampered due to the neutralization resistance associated with its envelope glycoprotein. We generated a MVA recombinant that expresses the less neutralization-resistant SIVsmE543-3 envelope and a plasmid of the codon-optimized gp140 (also SIVsmE543-3) that we used to generate recombinant trimeric envelope. A cohort of 24 rhesus macaques (6 per group) were immunized with either: A) 543 env trimer + AbISCO-100 adjuvant alone, B) MVA-nonrecombinant + adjuvant, C) Two MVA-Env primes, + two trimer boosts with adjuvant, and D) MVA-Env plus trimer co-administered 4 times with adjuvant. All SIV Env immunized animals developed neutralizing antibodies to SIVsmE660 but only weak and partial neutralization of SIVsmE543. They were challenged intrarectally with a high dose of SIVsmE660, 4 months following the last immunization, at a time when neutralizing antibody had waned to very low levels. A delay and blunting of viremia was observed in 50% of animals that received the prime boost regimen with one animal not apparently infected (Group C). However a macaque each from Groups B and D also remained uninfected so any uninfected animals are being rechallenged IR. Immunization-generated and anamnestic antibody responses are currently under study to determine correlates of the observed partial protection. Future studies will focus on the prime/boost protocol with challenge at peak immune responses (4 weeks after last immunization) using a repetitive, lower dose SIVsmE660 challenge model. We collaborated with a number of groups on vaccine studies using SIVsmE543 immunogens or SIVsmE660 challenge viruses including: 1) Dr. Barbara Felber and George Pavlakis at the NCI, 2) Dr. Harriet Robinson at Emory and 3) Dr. Dan Barouch (Harvard) and Nelson Michael (WRAIR). DEVELOPMENT OF ALTERNATIVE CHALLENGE STRAINS The development and characterization of alternative to the major SIVmac239/2251 and SIVsmE660 challenge strains are required for studies evaluating the role of neutralizing antibody in protection. We wish to develop and characterize additional strains of SIV that are more sensitive to neutralization and that can model the Tier2 type sensitivity of primary HIV isolates. One strain we have investigated is the minimally pathogenic, neutralization sensitive SIVsmH4i. This clone has a defective nef gene due to frameshift mutation in the C-terminus of this gene. We generated a corrected version of this clone, SIVsmH4i Nef+. The two clones produced similar levels of virus following transfection of 293T cells, but the correction restored expression of the Nef protein. Intravenous inoculation of macaques with SIVsmH4i resulted in delayed and low levels of peak viremia. This contrasted with robust peak viremia in macaques inoculated with the corrected version and viral kinetics similar to that observed in pathogenic SIV strains. Despite the restoration of in vivo replication ability, neither clone resulted in significant acute memory CD4+ T cell loss or disease in a period of two years of follow-up. We are also presently evaluating two full length infectious clones of SIVsmE660. These both replicate efficiently in macaque PBMC in vivo, are sensitive to neutralization and replicate efficiently in rhesus macaques in vivo. As described in the pathogenesis project (AI000370) these clones are still sensitive to TRIM restriction and recent studies from the Watkins and Letvin labs suggest that TRIM5 polymorphisms and in particular expression of the restrictive TRIM-TFP allele contributes to protection in low does SIVsmE660 challenge models. Therefore we have generated minimal point mutants in CA that are now resistant to TRIM restriction that will be evaluated in vivo. These clones or uncloned viruses generated from macaque passage would be ideal source of challenge strains that should avoid TRIM restriction as a confounding factor in low dose E660 challenge models.